An alternative strategy for inhibition of the stringent response is to take advantage of the intimate connection between the stringent response and ribosomal protein biosynthesis and to exploit existing antibiotics that target bacterial protein biosynthesis.The cyclic peptide thiostrepton is an efficient inhibitor of both translational GTPases, targeting initiation factor IF2 and elongation factors EF-Tu and EF-G on the ribosome (26–28), and Rel A (at least in the test tube [29, 30]).(p)pp Gpp synthetase Rel A is activated by deacylated t RNA in the ribosomal A-site.An increase in (p)pp Gpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)pp Gpp synthesis.However, like Spo T (and unlike Rel A), Rel is capable of hydrolyzing (p)pp Gpp (17).The “short” RSH proteins are single-domain proteins that either synthesize (small alarmone synthetase [SAS]) or degrade (small alarmone hydrolase [SAH]) (p)pp Gpp.An acute increase in (p)pp Gpp levels upon stress—the so-called stringent response—drives the reallocation of available metabolic resources, gearing up bacterial physiology for stress resistance and survival.
In response to stress conditions, the activity of SASs is regulated on the transcriptional level (10, 18), as well as via activation by (p)pp Gpp (19, 20).
We show that in a biochemical system from purified components, the antibiotic thiostrepton efficiently inhibits Rel A activation by the A-site t RNA.
In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)pp Gpp induced by the Ile-t RNA synthetase inhibitor mupirocin.
This antibiotic intercalates between helices 43 and 44 of 23S r RNA and the ribosomal protein L11 (31).
The latter is indispensable for the functionality of Rel A (32), while the activity of EF-G is only moderately affected by the removal of L11 (33).